Ribosomes are the complex, cellular machinery responsible for the production of all proteins in every living organism. This 2.5 million Dalton enzyme contains three large RNAs and more than 50 proteins that form two asymmetric subunits and promote mRNA-directed translation of the genetic code. Accurate translation requires the precise synchronization of regulatory factors, messenger RNAs and transfer RNAs to produce a mature protein. Errors associated with translation are detrimental to gene expression and hence cellular function. Furthermore, consistent with the critical importance of error- free protein synthesis for proper cellular function, there are numerous examples where human disease is linked to alterations in this macromolecular machinery that monitors the accuracy of these events. The major question that underlies translational regulation is how the ribosome is able to distinguish errors from non-canonical three-base decoding and tRNA misreading from normal function. Our long-term goal is to understand how this large macromolecular machine on a molecular level identifies such errors and how this process impacts human disease. This long-term goal will be addressed here by testing the hypothesis that mRNA and tRNA interactions with the ribosome cause conformational changes that prevent errors either through suppression of the mRNA mutation or via a new and novel proofreading mechanism for quality control purposes. Three independent but complementary aims are proposed. Experiments in Aim 1 will test if the +1 shift into the new frame is promoted by interactions with elongation factor G in the A site or by ribosomal components that structurally obstruct the tRNA path between the A and P site. In Aim 2 we will structurally characterize how fs tRNASufA6 interacts with the ribosomal P site and biochemically test whether mutations of important tRNA nts affect fs tRNAs affinity for the A site and/or how it is recognized by EF-G and moved from the A to the P site. These experiments build upon our new model for +1 frameshifting we established in the prior funding period. In Aim 3 we will investigate a second, possibly linked phenomenon: how mismatched P-site mRNA-tRNA interactions on the ribosome arising from tRNA selection errors lead to premature termination of protein synthesis. These aims will be accomplished through a combination of structural biology of large, functional ribosomal complexes using both X-ray crystallography and single particle cryo-electron microscopy (in collaboration with Dr. Skiniotis) approaches and complementary biochemical methods.